PROJECT SUMMARY/ABSTRACT Obesity has reached epidemic proportions in the U.S. and plays a major role in the development of type 2 diabetes, dyslipidemia, and cardiovascular disease. There remains a very significant need for better non- surgical treatments. While most current weight loss agents act by suppressing appetite, strategies that can safely enhance energy expenditure have the potential to effectively treat obesity. Brown adipose tissue (BAT) is a thermogenic tissue that uniquely expresses mitochondrial UnCoupling Protein-1 (UCP1). This protein dissipates, in a regulated fashion, the electrochemical gradient in the mitochondria of brown adipocytes as heat, and thus plays an important role in the maintenance of body temperature and energy balance in rodents and humans. BAT is a flexible tissue that normally enlarges or atrophies over time depending on environmental temperature. In many different rodent models, enhancement of BAT mass has convincingly been shown to lead to weight loss and diabetes resistance. While BAT was until recently thought to be effectively nonexistent in adult humans, data obtained in the past several years show that adults in fact have significant BAT and that this tissue is functional. It has been well established that a higher amount of active BAT in individuals is strongly correlated with leanness. Cold exposure in humans leads to increased BAT formation, thermogenesis, insulin sensitivity, and lipolysis, demonstrating that BAT can be recruited and lead to metabolic benefits. Moreover, the genetic locus most tightly linked with general obesity causes defective recruitment of new brown adipocytes. Until recently no brown adipocyte stem cell had been identified. We discovered human brown adipocyte progenitor cells resident in skeletal muscle that under appropriate conditions become fully functional brown adipocytes, with high levels of UCP1 and a very high metabolic rate. These cells are a unique tool that we used to develop an assay for identifying compounds with the capacity to recruit new BAT. We recently converted this assay into high throughput format and employed it to screen 7000+ compounds. We validated the screening hits, and have selected the most promising of these based on potency, maximal activity, and the potential to create improved analogs. In the proposed work, we aim to investigate the Structure Activity Relationship of the most preferred of these compounds by purchasing and synthesizing a series of novel analogs and evaluating them in vitro. We will identify those with the highest potential for advancement based on activity and physicochemical and ADME properties. A lead compound and backup will be selected, and if this work is successful we plan to advance to PK and in vivo efficacy studies. These will be followed by lead optimization, selection of a development candidate, and generation of IND-enabling safety data.